CN105949289B - Obtained from the flavanols modulin MsMYB12L and its encoding gene of functional form apple and application - Google Patents
Obtained from the flavanols modulin MsMYB12L and its encoding gene of functional form apple and application Download PDFInfo
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Abstract
The invention discloses a kind of obtained from the flavanols modulin MsMYB12L and its encoding gene of functional form apple and application.Protein provided by the invention is obtained from apple, is named as MsMYB12L albumen, is following (a1) or (a2): the protein that (a1) amino acid sequence shown in sequence 1 in sequence table forms；(a2) by the amino acid sequence of sequence 1 by the substitution and/or deletion and/or addition of one or several amino acid residues and the protein as derived from sequence 1 relevant to plant flavanols compounds content.The gene (MsMYB12L gene) for encoding the MsMYB12L albumen also belongs to protection scope of the present invention.It present invention finds MsMYB12L albumen and its function, can be used for cultivating the genetically modified plants of flavanols compounds content change, there is major application prospect in plant breeding.
Description
Technical field
The present invention relates to a kind of flavanols modulin MsMYB12L and its encoding gene obtained from functional form apple and answer
With.
Background technique
" doctor's food homology " is developing direction.Apple storage property is good, and supply cycle is long, is worldwide fruit, and especially fruit contains
Have higher proportion, human body be easier absorb free polyphenol, have well it is anti-oxidant, antitumor, prevention cardiovascular and cerebrovascular
The effects of disease and liver protection, healthy nutritive value is high, there is " one day apple, doctor is far from me " (An apple a day
keeps the doctor away！) good reputation, in the world considerable country be all classified as major consumers fruit and energetically
Recommend.But finding in recent years shows, on the one hand, a apple variety more than 1000 be bred as both at home and abroad in the past few decades,
80% it is the hybridization of kinds such as ' Jin Shuai ', grows directly from seeds or bud selection offspring, this " inbreeding " often bring the hereditary basis of kind
The problems such as narrow and resistance declines；On the other hand, characteristic, more anti-and diversity fruits become the important directions of industry development.
Malus sieversii and its red meat modification (Malus sieversii f.neidzwetzkyana) are world's cultivation apples
Ancestors' kind of fruit, not only genetic diversity is extremely abundant, but also rich in functions, health-care components such as flavonoids, be carry out it is degeneration-resistant with
The precious gene pool of quality breeding.But because reasons, the genetic diversities of Malus sieversii such as the farmland reclamation of wasteland are just seriously damaged,
It is endangered；China is maximum apple production and country of consumption in the world, wherein 39,500,000 tons of apple of production in 2012, main to use
In fresh food, and nearly 70% is the lower Fuji's kind of Flavonoid Content.
Therefore, around " scientific conservation of Malus sieversii resource is opened up with Sustainable and highly-efficient use, cultivar hereditary basis
Exhibition, Apple Industry transition and upgrade are promoted to side structure reform and Practices Sustainable Increasing Income of Farmers and level of human health together ", joined
Tackling key problem and integration and demonstration are closed, the present inventor and the professor of Cornell Univ USA cooperate, to Malus sieversii and Europe
Worldwide 97 parts of apple resources such as forest apple have carried out genome and have resurveyed sequence and bioinformatic analysis, construct new
Boundary red meat apple and the apple variety first generation of hybrid and one, two generation segregating populations of backcrossing, research specify that Malus sieversii group loses
Pass the hereditary variation feature of the characters such as technical parameter, the Flavonoid Content that structure and genetic diversity feature, Core Germplasms construct
And development mechanism, propose " functional form apple " concept and " wide row high level cadre, inter-row green covering, to grass fertilising, fertilize the soil support root "
Modern orchard management philosophy creates the apple high-efficient breeding technique system of conventional hybridization and biotechnology combination, initiative
A collection of new varieties and excellent germplasm, have developed Variety of Apple highly effective matched cultivation technical system.Currently, having authorized and having declared
10 remainder of patent of invention is colonized more than 40,000 strain of hybrid seedling, incubation new varieties (being) 16；Deliver correlative study paper 120
, wherein more than 20 piece of SCI paper, these research achievements are totally in the top standard of international similar research.
Summary of the invention
The object of the present invention is to provide a kind of flavanols modulin MsMYB12L and its coding obtained from functional form apple
Gene and application.
Protein provided by the invention is obtained from apple, is named as MsMYB12L albumen, is following (a1) or (a2):
(a1) protein that the amino acid sequence shown in sequence 1 in sequence table forms；
(a2) amino acid sequence of sequence 1 by the substitution of one or several amino acid residues and/or missing and/or is added
Add and the protein as derived from sequence 1 relevant to plant flavanols compounds content.
In order to make MsMYB12L albumen in (a) convenient for purifying and detection, can in as sequence table amino shown in sequence 1
The amino terminal or carboxyl terminal of the protein of acid sequence composition connect upper label as shown in Table 1.
The sequence of 1 label of table
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
MsMYB12L albumen in above-mentioned (b) can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological expression
It obtains.The encoding gene of MsMYB12L albumen in above-mentioned (b) can be by will lack in DNA sequence dna shown in sequence 2 in sequence table
The codon of one or several amino acid residues is lost, and/or carries out the missense mutation of one or several base-pairs, and/or at it
The coded sequence that 5 ' ends and/or 3 ' ends connect label shown in table 1 obtains.
The gene (MsMYB12L gene) for encoding the MsMYB12L albumen also belongs to protection scope of the present invention.
The gene is following (1) or (2) or (3):
(1) DNA molecular shown in sequence 2 in coding region sequence table；
(2) hybridize and encode and plant flavanols compounds content phase with the DNA sequence dna that (1) limits under strict conditions
The DNA molecular of the protein of pass；
(3) DNA sequence dna limited with (1) has 90% or more homology and encodes and plant flavanols compounds content
The DNA molecular of relevant protein.
Above-mentioned stringent condition can be for 0.1 × SSPE (or 0.1 × SSC), the solution of 0.1%SDS be miscellaneous in DNA or RNA
It hands over and hybridizes at 65 DEG C in experiment and wash film.
Recombinant expression carrier, expression cassette, transgenic cell line, Transgenic plant tissue containing the MsMYB12L gene
Or recombinant bacterium all belongs to the scope of protection of the present invention.
The recombinant expression carrier of MsMYB12L gene can be contained with existing plant expression vector construction.The plant expression
Carrier includes double base agrobacterium vector and the carrier etc. that can be used for plant micropellet bombardment.Use the gene constructed recombination table of MsMYB12L
When up to carrier, it can be opened before its transcription initiation nucleotide plus any enhanced, composing type, organizing specific type or induction type
Mover, they can be used alone or are used in combination with other plant promoters；In addition, using the gene constructed recombination of MsMYB12L
When expression vector, also enhancer, including translational enhancer or transcriptional enhancer, these enhancer regions can be used to can be ATG
Initiation codon or neighboring region initiation codon etc., but must be identical as the reading frame of coded sequence, to guarantee entire sequence
Correct translation.The source of the translation control signal and initiation codon be it is extensive, can be natural, be also possible to close
At.Translation initiation region can come from transcription initiation region or structural gene.For the ease of to transgenic plant cells or plant
Object is identified and is screened, and can be processed to plant expression vector used, and the expression in plant, which is such as added, can produce color change
The enzyme of change or gene, resistant antibiotic marker or the anti-chemical reagent marker gene of luminophor etc..From turn
Any selected marker can be not added in the security consideration of gene plant, directly with phenotypic screen transformed plant.The recombination
The carrier that sets out of expression vector can be pRI101 carrier.The recombinant expression carrier is concretely in the Nde I of pRI101 carrier
The recombinant plasmid pRI101- that double chain DNA molecule shown in the sequence 2 of insetion sequence table obtains between BamH I restriction enzyme site
MsMYB12L。
The plant tissue concretely plant callus, more specifically can be plant embryo callus.The plant
It can be monocotyledon or dicotyledon.The dicotyledon concretely rosaceous plant.The rosaceous plant tool
Body can be Malus.The Malus concretely apple, more specifically can be ' Wang Lin ' apple.
The present invention also protects the application of the MsMYB12L albumen, for as follows (b1) or (b2):
(b1) regulate and control the flavanols compounds content of plant；
(b2) increase the flavanols compounds content of plant.
The plant can be monocotyledon or dicotyledon.The dicotyledon concretely rosaceous plant.
The rosaceous plant concretely Malus.The Malus concretely apple more specifically can be ' Wang Lin '
Apple.
The present invention also protects the MsMYB12L gene cultivating the increased genetically modified plants of flavanols compounds content
In application.The plant can be monocotyledon or dicotyledon.The dicotyledon concretely rosaceous plant.
The rosaceous plant concretely Malus.The Malus concretely apple more specifically can be ' Wang Lin '
Apple.
The present invention also protects a kind of method for cultivating genetically modified plants, includes the following steps: the MsMYB12L gene
Importing is set out plant, and the genetically modified plants that flavanols compounds content is higher than the plant that sets out are obtained.The plant that sets out
It can be monocotyledon or dicotyledon.The dicotyledon concretely rosaceous plant.The rosaceous plant tool
Body can be Malus.The Malus concretely apple, more specifically can be ' Wang Lin ' apple.The MsMYB12L
Gene can specifically pass through the plant that sets out described in the importing of any description above recombinant expression carrier.It, specifically can be by institute in the method
It states MsMYB12L channel genes to set out the callus of plant, then cultivates callus for plant.The callus tool
Body can be embryo callus.The recombinant expression carrier for carrying the MsMYB12L gene can be by Ti-plasmids, Ri plasmid, plant
The conventional biology methods such as object viral vectors, directly delivered DNA, microinjection, conductance, mediated by agriculture bacillus are transformed into plant cell
Or in tissue.
The present invention also protects a kind of method for obtaining Transgenic plant tissue, includes the following steps: the MsMYB12L
Channel genes set out plant tissue, obtain the genetically modified plants group that flavanols compounds content is higher than the plant tissue that sets out
It knits.The plant that sets out can be monocotyledon or dicotyledon.The dicotyledon concretely rosaceous plant.Institute
State rosaceous plant concretely Malus.The Malus concretely apple, more specifically can be ' Wang Lin ' apple
Fruit.The MsMYB12L gene can specifically pass through the plant tissue that sets out described in the importing of any description above recombinant expression carrier.Institute
State plant tissue concretely plant callus.The callus concretely embryo callus.
The present invention also protects the MsMYB12L albumen or the MsMYB12L gene or any description above method
Application in plant breeding.The purpose of the plant breeding is the plant cultivating flavanols compounds content and increasing.
Any description above flavanols compounds are the compound with female ring shown in formula (I)；
Any description above flavanols compounds are compound shown in formula (II)；
R1=H, OH or OG；
R2=OH or H；
R3=H or OH.
R1=H, R2=OH, R3It is catechin when=H
R1=OH, R2=H, R3It is epicatechin when=H
R1=H, R2=OH, R3It is nutgall catechin when=OH
R1=OH, R2=H, R3It is epigallocatechin when=OH
R1=OG, R2=H, R3It is L-Epicatechin gallate when=H
R1=OG, R2=H, R3It is Epigallo-catechin gallate (EGCG) when=OH
Present invention finds MsMYB12L albumen and its function, it can be used for cultivating turning for flavanols compounds content change
Gene plant has major application prospect in plant breeding.
Detailed description of the invention
Fig. 1 is photo before DMACA is dyed.
Fig. 2 is photo after DMACA dyeing.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
PRI101 carrier (also known as " pRI101-AN DNA "): Takala company, Code No.3262.Agrobacterium
LBA4404:Tiangen company, catalog number: CC2901.' Wang Lin ' apple: bibliography: " The bHLH
transcription factor MdbHLH3 promotes anthocyanin accumulation and fruit
colouration in response to low temperature in apples》。
The discovery of embodiment 1, MsMYB12L albumen and its encoding gene
Have found that a new albumen is named as shown in the sequence 1 of sequence table from " purplish red No. 1 " apple
MsMYB12L albumen.It is MsMYB12L gene, open reading frame such as sequence table by the unnamed gene for encoding MsMYB12L albumen
Sequence 2 shown in.
In GENBANK, with the highest albumen of 1 homology of sequence as shown in the sequence 3 of sequence table.By the sequence 3 of sequence table
Shown in protein be named as reference protein, encoding gene is named as crt gene, as shown in the sequence 4 of sequence table.
The Function Identification of embodiment 2, MsMYB12L albumen
One, construction recombination plasmid
The Nde I and BamH of the insertion pRI101 carrier of double chain DNA molecule shown in sequence 2 by artificial synthesized sequence table
Between I restriction enzyme site, recombinant plasmid pRI101-MsMYB12L is obtained.
The Nde I and BamH of the insertion pRI101 carrier of double chain DNA molecule shown in sequence 4 by artificial synthesized sequence table
Between I restriction enzyme site, control plasmid is obtained.
Two, the acquisition of genetically modified organism
1, recombinant plasmid pRI101-MsMYB12L is imported into Agrobacterium LBA4404, obtains recombinational agrobacterium.
2, the recombinational agrobacterium thallus for taking step 1 to obtain is suspended with 30ml MS fluid nutrient medium, obtains OD600nm=0.8
Bacteria suspension, be added 30 μ L acetosyringones, obtain infected liquid.
3, the embryo callus for taking ' Wang Lin ' apple is seeded on MS solid medium, and 25 DEG C are cultivated 15 days.
4, after completing step 3, callus is taken, is immersed in the infected liquid that step 2 obtains, 160rpm shaken at room temperature 15-
Then 20min blots surface with sterilizing filter paper.
5, after completing step 4, callus is taken, is placed in co-culture medium (containing 1mg/L 2,4-D and 0.5mg/L6-BA
MS solid medium) on, 25 DEG C dark culturing 36 hours.
6, after completing step 5, callus is taken, screening and culturing medium (kanamycins containing 50mg/L, 250mg/L carboxylic Bian are placed in
Penicillin, 1mg/L 2, the MS solid medium of 4-D and 0.5mg/L 6-BA) on, 25 DEG C dark culturing 20-30 days.
7, after completing step 6, the resistant calli that can be grown on screening and culturing medium is taken, carries out PCR identification.
PCR identification primer pair used is as follows:
35s-F:5 '-GACGCACAATCCCACTATCC-3 '；
MYB12L-R:5 '-GACTACTCTCTCCAACTC-3 '；
35s-F corresponds to the section in the 35S promoter on carrier framework, and MYB12L-R corresponds in MsMYB12L gene
Section, target sequence length is about 1000bp.
The positive callus of PCR identification is to turn MsMYB12L gene callus.
8, the MsMYB12L gene callus that turns for obtaining step 7 is seeded to screening and culturing medium (that is mould for card containing 50mg/L
The MS solid medium of element, 250mg/L carboxylic Bian penicillin, 1mg/L 2,4-D and 0.5mg/L 6-BA) on carry out squamous subculture.
Three, the acquisition of control tissue
It replaces recombinant plasmid pRI101-MsMYB12L to carry out step 2 control plasmid, obtains turning crt gene callus group
It knits.
It replaces recombinant plasmid pRI101-MsMYB12L to carry out step 2 in pRI101 carrier, obtains turning empty carrier callus group
It knits.
Four, the identification organized
1, Qualitative Identification (DMACA decoration method)
DMACA decoration method principle: in acid condition, can be with flavanol compound to dimethylaminocinnamaldehyde (DMACA)
Closing object, (flavanols compounds include three classes: the first kind is natural flavan-3-alcohol class；Second class is flavane -3,4- glycols,
That is leucoanthocyanidin；Third class is procyanidine) colour developing generation blue, color and concentration are in positive relationship.Bibliography:
" optimization of tea root procyanidine extraction process and detection method ".
Callus to be measured, which is respectively as follows:, to be turned MsMYB12L gene callus, turns empty carrier callus and Colombia
Arabidopsis thaliana ecotype callus.
It is operated in accordance with the following steps:
(1) 1 parts by volume methanol and 1 parts by volume 6M HCL aqueous solution are mixed, obtains methanol-hydrochloric acid mixed solution.
(2) DMACA is dissolved in methanol-hydrochloric acid mixed solution, obtains the DMACA solution of 0.3g/100ml.
(3) callus to be measured is taken, dyes 15min in DMACA solution.
Photo is shown in Fig. 1 before DMACA is dyed.Turning MsMYB12L gene callus is buff.Columbia ecotype is quasi-
Southern mustard callus and to turn empty carrier callus be light yellow.
Photo after DMACA dyeing is shown in Fig. 2.Turn MsMYB12L gene callus and navy blue dyed by DMACA, it is meant that
There is the accumulation of a large amount of flavanols compounds.Columbia ecotype Arabidopsis callus and turn empty carrier callus not
It is dyed to navy blue, is shown as lightpink.The result shows that MsMYB12L albumen can regulate and control the synthesis of flavanols compounds.
It carries out five repetitions to test, as a result unanimously.
2, Quantitative measurement
Callus to be measured, which is respectively as follows:, to be turned MsMYB12L gene callus, turns crt gene callus, turns empty carrier
Callus and Columbia ecotype Arabidopsis callus.
It is operated in accordance with the following steps:
(1) 0.2g callus to be measured is taken, is transferred in centrifuge tube after liquid nitrogen grinding.
(2) 70% (volumn concentration) of 1ml ascorbic acid containing 1mg/ml is added in the centrifuge tube for taking step (1) to obtain
Aqueous acetone solution, collects supernatant, remaining precipitating in centrifuge tube by 4 DEG C, stand under dark condition and extract 30min.
(3) 70% (volumn concentration) of 1ml ascorbic acid containing 1mg/ml is added in the centrifuge tube for taking step (2) to obtain
Aqueous acetone solution, collects supernatant, remaining precipitating in centrifuge tube by 4 DEG C, stand under dark condition and extract 30min.
(4) 70% (volumn concentration) of 1ml ascorbic acid containing 1mg/ml is added in the centrifuge tube for taking step (5) to obtain
Aqueous acetone solution, collects supernatant, remaining precipitating in centrifuge tube by 4 DEG C, stand under dark condition and extract 30min.
(5) 70% (volumn concentration) of 1ml ascorbic acid containing 1mg/ml is added in the centrifuge tube for taking step (4) to obtain
Aqueous acetone solution, collects supernatant by 4 DEG C, stand under dark condition and extract 10 hours.
(6) supernatant and step obtained supernatant that supernatant that step (2) obtains, step (3) obtain, step (4)
Suddenly the supernatant mixing that (5) obtain, obtains mixed liquor.
(7) mixed liquor for taking step (6) to obtain, is added 3ml ether, and -20 DEG C, stand that (split-phase, upper layer are under dark condition
Ether phase, lower layer are acetone phase, and flavanols compounds are mutually contained in lower layer), collect lower layer's phase.
(8) the lower layer's phase for taking 2ml step (7) to obtain, the DMACA that (2) preparation of 1ml methanol and 0.5ml step 1 is added are molten
Liquid is stored at room temperature 20min, and then 643nm measures absorbance.
Standard curve is made with (+)-Catechin (sigma product), calibration curve equation is as follows: y=2.816x-
0.012, R2=0.996 (y: light absorption value；X: flavanols compounds concentration, unit mg/g).
Absorbance is brought into calibration curve equation, to calculate the flavanols compounds content in seed to be measured.
It carries out five repetitions to test, repeats the average value for taking 10 parts of callus to be measured in test every time.
The flavanols compounds content for turning MsMYB12L gene callus is 124.562mg/kg.Turn crt gene to be cured
The flavanols compounds content of injured tissue is 63.254mg/kg.Turn the flavanols compounds content of empty carrier callus
For 21.758mg/kg.The flavanols compounds content of Columbia ecotype Arabidopsis callus is 21.604mg/kg.
What " kg " in this section referred to is callus fresh weight.
Claims (10)
- The application of 1.MsMYB12L albumen, for the flavanols compounds content for increasing plant；The MsMYB12L albumen be by The protein that amino acid sequence shown in sequence 1 forms in sequence table.
- 2. the gene for encoding MsMYB12L albumen is cultivating the application in the increased genetically modified plants of flavanols compounds content； The protein that MsMYB12L albumen amino acid sequence shown in sequence 1 in sequence table forms.
- 3. application as claimed in claim 2, it is characterised in that: the gene of the coding MsMYB12L albumen is code area such as sequence DNA molecular shown in sequence 2 in list.
- 4. a kind of method for cultivating genetically modified plants includes the following steps: to set out the channel genes for encoding MsMYB12L albumen Plant obtains the genetically modified plants that flavanols compounds content is higher than the plant that sets out；The MsMYB12L albumen be by The protein that amino acid sequence shown in sequence 1 forms in sequence table.
- 5. method as claimed in claim 4, it is characterised in that: the gene of the coding MsMYB12L albumen is code area such as sequence DNA molecular shown in sequence 2 in list.
- 6. a kind of method for cultivating Transgenic plant tissue, includes the following steps: that the channel genes of MsMYB12L albumen will be encoded Set out plant tissue, obtains the Transgenic plant tissue that flavanols compounds content is higher than the plant tissue that sets out；It is described The protein that MsMYB12L albumen amino acid sequence shown in sequence 1 in sequence table forms.
- 7. method as claimed in claim 6, it is characterised in that: the gene of the coding MsMYB12L albumen is code area such as sequence DNA molecular shown in sequence 2 in list.
- 8. method as claimed in claim 6, it is characterised in that: the plant tissue is plant callus.
- 9.MsMYB12L albumen, or, the gene of coding MsMYB12L albumen, or, claim 4 or 5 or 6 or 7 or 8 sides Method, the application in plant breeding；What MsMYB12L albumen amino acid sequence shown in sequence 1 in sequence table formed Protein.
- 10. application as claimed in claim 9, it is characterised in that: the gene of the coding MsMYB12L albumen be code area such as DNA molecular shown in sequence 2 in sequence table.
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| CN109295069B (en) * | 2018-09-19 | 2021-08-20 | 昆明理工大学 | Application of rhizoma panacis majoris transcription factor gene PjMYB1 |
| CN113461794B (en) * | 2021-08-19 | 2022-05-03 | 云南省烟草农业科学研究院 | Kit and method for regulating seed germination and application thereof |
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2016
- 2016-05-06 CN CN201610300919.1A patent/CN105949289B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| Accession NO.: XP_008337875.1，PREDICTED: transcription factor MYB12 isoform X2 [Malus domestica];None;《Genbank Database》;20140620;DEFINITION、SOURCE、FEATURES及ORIGIN部分 |
| Different coloration patterns between the red- and white-fleshedfruits of malus crabapples;Ya-Ru Wang等;《Scientia Horticulturae》;20150808;第194卷;第26-33页 |
| Transient expression vectors for functional genomics,;Roger P Hellens等;《Plant Methods》;20051218;第1卷;第1-14页 |
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